Hematopoietic stem cells (HSC) have served as a fundamental model for the study of stem cell biology and as a vital therapeutic modality for the treatment of hematopoietic malignancies and bone marrow failure syndromes. Although multiple signaling pathways are implicated in the regulation of HSCs, how these signals are integrated at the level of the HSC to maintain the balance between self-renewal and lineage commitment remains a major question in HSC research. Our recent work has demonstrated that the signaling molecule glycogen synthase kinase-3 (GSK-3) plays a pivotal role in HSC homeostasis in vivo. Pharmacological and genetic loss of function studies reveal that GSK-3 suppresses two, distinct pathways in hematopoietic stem/progenitor cells (HSPCs), the Wnt/ss-catenin and mTOR pathways. Inhibition of GSK-3 activates both pathways in HSPCs-with distinct consequences: activation of Wnt signaling causes expansion of phenotypic HSPCs, whereas mTOR activation causes HSPC exhaustion. Thus, combined inhibition of GSK-3 and mTOR yields a net expansion of HSPCs in vivo, as Wnt-stimulated HSPC expansion is no longer balanced by mTOR-mediated HSPC depletion. These observations demonstrate that GSK-3 is an important regulator of HSC homeostasis and suggest a therapeutic strategy to expand HSCs in vivo and ex vivo using currently available FDA-approved agents, including lithium to inhibit GSK-3 and rapamycin/sirolimus to inhibit mTOR. Our preliminary data further indicate that this drug combination may allow long-term culture of mouse HSCs ex vivo in the absence of hematopoietic cytokines. We will therefore test whether simultaneous inhibition of GSK-3 and mTOR expands functional HSCs under both in vivo and ex vivo conditions. If successful, this project will define a critical regulatory node in HSC homeostasis and will identify approaches to expand HSCs for improved HSCT therapy. PUBLIC HEALTH RELEVANCE: This proposal investigates the regulation of blood forming cells and specifically how the body maintains the ability to produce a high volume of mature blood cells and at the same time maintains the population of cells in the bone marrow needed for new blood formation. The proposal explores new approaches to enhance blood formation for bone marrow transplantation and in patients with low blood counts due to disease or chemotherapy.